Acute otitis media is a persisting health problem among children in this country and elsewhere. Nontypable Haemophilus influenzae are a major cause of this illness. The objective of this proposal is to gain additional understanding of the host factors important in protective immunity against otitis media caused by this organism. In Aim I will define the importance of antibody to a 39 Kd major outer membrane protein of nontypable Haemophilus influenzae in mediating protective immunity in the chinchila otitis model. Both convalescent sera from animals recovered from infection and immune sera which are protective in passive protection experiments contain high levels of antibody against the 39 Kd protein of the homologous nontypable Haemophilus strain. Affinity-purified or nonclonal antibodies will be prepared against the 39 Kd proteins of several prototype strains and assayed in passive protection experiments. Purified 39 Kd proteins from prototype strains will be tested as vaccine candidates in the animal model. In Aim 2, I will define strain differences in the 39 Kd proteins of nontypable Haemophilus as they relate to immunity to otitis. Affinity-purified or monclonal antibody preparations recognizing the 39 Kd proteins will be tested against a panel of nontypable strains in fluorescent antibody, whole cell radioimmunoprecipitation and bactericidal assays. In vitro studies will be followed with preliminary in vivo cross-protection assays. In Aim 3, I will define the cell surface antigens of nontypable Haemophilus which stimulate serum and middle ear fluid antibody responses in the course of human infection. Acute and convalescent samples will be assayed with radioimmunoprecipitation, ELISA, and bactericidal assays. Absorption experiments will be performed with purified outer membrane proteins and lipopolysaccharide in an effort to identify targets of bactericidal antibody. The information derived from this work should permit us to better assess whether vaccination is a feasible strategy to pursue for the prevention of nontypable Haemophilus otitis media.